ΒETA-LACTAMASES BEYOND THE BACTERIAL CELL: SELECTIVE TRANSPORT MEDIATED BY OUTER MEMBRANE VESICLES

LÓPEZ, CAROLINA; PRUNOTTO, ALESSIO; BAHR, GUILLERMO; CAPODIMONTE, LUCIA; GONZÁLEZ, LISANDRO; DAL PERARO, MATTEO; VILA, ALEJANDRO

Los Cocos, Córdoba

Congreso; XVII Congreso Argentino de Microbiología General; 2022

Sociedad Argentina de Microbiología General (SAMIGE)

COMUNICACÓN ORALOuter membrane vesicles (OMVs) are spherical lipid bilayer nanostructures released by Gramnegative bacteria into the extracellular milieu. OMVs act as carriers of resistance determinants such as β-lactamases and related plasmids. OMVs incorporating β-lactamases can protect populations of susceptible bacteria against antibiotics, as recently shown in vivo, and have been postulated as a novel mechanism of plasmid transfer. In particular, Metallo-β-lactamases (MBLs) are Zn(II)-dependent β- lactamases, that represent one of the largest groups of carbapenemases for which clinical inhibitors are not yet commercially available. Among MBLs, NDMs, VIMs and IMPs are the enzymes with the highest clinical relevance and geographical dissemination. We demonstrated that some MBLs can be packed into OMVs. The encapsulation of MBLs into vesicles may be due to a detoxification mechanism in response to the stress imposed on the bacterial host by the expression of certain MBLs. While the transport of toxic species is non-specific, the packaging of folded and active MBLs is selective. However, the features that govern this selective transport mediated by OMVs are largely unknown. Liposome flotation assays, mutagenesis and molecular dynamics (MD) simulations were used to identify the interactions between the MBLs and the bacterial membrane that play a relevant role in determining the amount of active MBLs present in Escherichia coli vesicles. OMVs were purified from E. coli expressing NDM-1, IMP-1, VIM-2 and their variants and protein levels were determined by immunodetection. We demonstrated that the membrane-anchored enzyme NDM-1 is packaged into outer membrane vesicles in an active form because of its cellular localization. However, a soluble variant of NDM-1 is packaged in smaller amounts, indicating that membrane anchoring is not the only molecular feature determining cargo selection. In addition to the lipid anchor, a positively charged patch on the surface of the soluble domain of NDM-1 makes attractive electrostatic interactions (mediated by two Arg residues) with the bacterial membrane and favors its inclusion into vesicles. Regarding natural soluble MBLs, IMP-1 was much more efficiently transported into OMVs than VIM-2. We demonstrated that a positively charged patch in the surface of IMP-1 (mediated by four Lys residues) favors its inclusion into OMVs. These residues are conserved among NDM and IMP enzymes, suggesting that the phenomenon is common to most allelic variants from these families. Instead, the lack of electrostatic interactions of VIM-2 with the membrane accounts for the small levels of this protein into OMVs. This reveals that some MBLs are tuned to interact with the bacterial membrane and therefore are selectively exported into OMVs in an active form